The present invention relates to the introduction of destabilizing moieties into oligonucleotide probes for the improvement of nucleic acid amplification processes, methods comprising the use of such oligonucleotide and to kits for performing nucleic acid amplification processes comprising such oligonucleotide probes. The present invention is particularly concerned with amplification of hybridised modified nucleic acid probes such that sensitivity and specificity of the reaction is increased.
All publications mentioned in the present specification are herein incorporated by reference.
A number of nucleic acid amplification processes are cited in the literature and disclosed in published European and PCT patent applications. One such process known as polymerase chain reaction (PCR) is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202. The PCR process consists of nucleic acid primers that anneal to opposite strands of a DNA duplex; these primers are extended using thermostable DNA polymerase in the presence of nucleotide triphosphates to yield two duplex copies of the original nucleic acid sequence. Successive cycles of denaturation, annealing and extension are undertaken to further amplify copies of the original nucleic acid sequence. This method has its drawbacks including the need for adjusting reaction temperatures alternately between intermediate (e.g. 50xc2x0 C.-55xc2x0 C.) and high (e.g. 90xc2x0 C.-95xc2x0 C.) temperatures involving repeated thermal cycling. Also the time scale required for multiple cycles of large temperature transitions to achieve amplification of a nucleic acid sequence and the occurrence of sequence errors in the amplified copies of the nucleic acid sequence is a major disadvantage as errors occur during multiple copying of long sequence tracts. Additionally, detection of the amplified nucleic acid sequence generally requires further processes e.g. agarose gel electrophoresis.
Alternative nucleic acid amplification processes are disclosed WO 88/10315 (Siska Diagnostics). EP 329,822 (Cangene) and 373,960 (Siska Diagnostics). U.S. Pat. No. 5,554,516 (Gen-Probe Inc.), and WO 89/1050 and 88/10315 assigned to Burg et al. and Gingeras et al., respectively. These amplification processes describe a cycling reaction comprising of alternate DNA and RNA synthesis. This alternate RNA/DNA synthesis is achieved principally through the annealing of oligonucleotides adjacent to a specific DNA sequence whereby these oligonucleotides comprise a transcriptional promoter. The RNA copies of the specific sequence so produced, or alternatively an input sample comprising a specific RNA sequence (U.S. Pat. No. 5,554,516), are then copied as DNA strands using a nucleic acid primer and the RNA from the resulting DNA:RNA hybrid is either removed by denaturation (WO 88/10315) or removed with RNase H (EP 329822, EP 373960 and U.S. Pat. No. 5,554,516). The annealing of oligonucleotides forming a transcription promoter is then repeated in order to repeat RNA production.
Amplification is thus achieved principally through the use of efficient RNA polymerases to produce an excess of RNA copies over DNA templates. The RNase version of this method has great advantages over PCR in that amplification can potentially be achieved at a single temperature (i.e. isothermally). Additionally, a much greater level of amplification can be achieved than for PCR i.e. a doubling of DNA copies per cycle for PCR, compared to 10-100 RNA copies using T7 RNA polymerase. A disadvantage associated with the DNA:RNA cycling method described in EP 329822 is that it requires test nucleic acid with discrete ends for the annealing of oligonucleotides to create the transcriptional promoter. This poses difficulties in detection of, for example, specific genes in long DNA molecules. Further disadvantages of this method are that at least three enzymes are required to undertake the DNA:RNA cycling with potentially deleterious consequences for stability, cost and reproducibility; and that one or more further processes are often required (e.g. gel electrophoresis) for detection of the amplified nucleic acid sequence.
The processes described above all refer to methods whereby a specific nucleic acid region is directly copied and these nucleic acid copies are further copied to achieve amplification. The variability between various nucleic acid sequences is such that the rates of amplification between different sequences by the same process are likely to differ thus presenting problems for example in the quantitation of the original amount of specific nucleic acid.
The processes listed above have a number of disadvantages in the amplification of their target nucleic acid; therefore, a list of desiderata for the sensitive detection of a specific target nucleic acid sequence is outlined below:
a) the process should preferably not require copying of the target sequence;
b) the process should preferably not involve multiple copying of long tracts of sequence;
c) the process should preferably be generally applicable to both DNA and RNA target sequences including specific sequences without discrete ends;
d) the signal should preferably result from the independent hybridisation of two different probes; or regions of probe, to a target sequence; and
e) the process should include an option for detection of hybridised probe without any additional processes.
A nucleic acid amplification process that fulfils the above desiderata is disclosed in WO 93/06240 (Cytocell Ltd). Two amplification processes are described, one thermal and one isothermal. Both the thermal and isothermal versions depend on the hybridisation of two nucleic acid probes of which regions are complementary to the target nucleic acid. Portions of said probes are capable of hybridising to the sequence of interest such that the probes are adjacent or substantially adjacent to one another, so as to enable complementary xe2x80x9carmxe2x80x9d specific sequences of the first and second probes to become annealed to each other. Following annealing, chain extension of one of the probes is achieved by using part of the other probe as a template.
Amplification is achieved by one of two means; in the thermal cycling version thermal separation of the extended first probe is carried out to allow hybridisation of a further probe, substantially complementary to part of the newly synthesised sequence of the extended first probe. Extension of the further probe by use of an appropriate polymerase using the extended first probe as a template is achieved. Thermal separation of the extended first and further probe products allows these molecules to act as a template for the extension of further first probe molecules and the extended first probe can act as a template for the extension of other further probe molecules. In the isothermal version, primer extension of the first probe creates a functional RNA polymerase promoter that in the presence of a relevant RNA polymerase transcribes multiple copies of RNA. The resulting RNA is further amplified as a result of the interaction of complementary DNA oligonucleotides containing further RNA polymerase promoter sequences, whereupon annealing of the RNA on the DNA oligonucleotide and a subsequent extension reaction leads to a further round of RNA synthesis. This cyclical process generates large yields of RNA, detection of which can be achieved by a number of means. The present invention is related to these processes and aims to provide improvements thereon.
In preferred embodiments the present invention also fulfils all the aforementioned desiderata. This may be achieved through the hybridisation of two oligonucleotide probes that contain complementary target specific regions together with complementary arm regions, such that in the presence of the target sequence of interest the target and the two probes form a xe2x80x9cthree way junctionxe2x80x9d. Within the complementary arm region of one or both of the oligonucleotide probes is incorporated a destabilizing moiety that prevents the two oligonucleotide probes from associating in the absence of target nucleic acid and hence reducing noise from the potential association of these probes.
In a first aspect the invention provides a pair of nucleic acid probes for use in a method of detecting a nucleic acid target sequence of interest, a first probe comprising a portion complementary to the sequence of interest and so capable of hybridising thereto and a portion non-complementary to the sequence of interest, and a second probe comprising a portion complementary to the sequence of interest and so capable of hybridising thereto and a portion non-complementary to the sequence of interest but complementary to that portion of the first probe which is non-complementary to the sequence of interest, such that the first and second probes are capable of hybridising to the sequence of interest in an adjacent or substantially adjacent manner so as to allow complementary portions of the first and second probes to hybridise to each other, characterised in that the first and/or second probe comprises a destabilizing, moiety which cannot base pair with the reciprocal member of the pair of probes, thereby preventing hybridisation of the first and second probes in the absence of the sequence of interest.
The target strand may comprise any nucleic acid (RNA or, more preferably DNA) sequence of interest, such as a sequence from a pathogen (such that the complex may be used to detect the presence of a pathogen), or may be the sequence of a particular human, animal or plant allele, such that the genotype of an individual human or animal may be determined. Conveniently (but not necessarily) at least that portion (typically 2-4 bases) of the target which contains the part of the second strand of the double stranded promoter will preferably comprise DNA. The target strand may comprise both DNA and/or RNA.
The hybridisation of the first and second probes to each other and to the sequence of interest forms a structure which the present inventors describe as a xe2x80x9cthree way junctionxe2x80x9d. The first and second probes preferably comprise DNA, PNA (peptide nucleic acid) or LNA (xe2x80x9clocked nucleic acidxe2x80x9d), but may comprise RNA, or any combination of the foregoing.
PNA is a synthetic nucleic acid analogue in which the sugar/phosphate backbone is replaced by a peptide-linked chain (typically of repeated N-(2-aminoethyl)-glycine units), to which the bases are joined by methylene carbonyl linkages. PNA/DNA hybrids have high Tm values compared to double stranded DNA molecules since in DNA the highly negatively-charged phosphate backbone causes electrostatic repulsion between the respective strands, whilst the backbone of PNA is uncharged. Another characteristic of PNA is that a single base mis-match is, relatively speaking, more destabilizing than a single base mis-match in heteroduplex DNA. Accordingly, PNA may advantageously be included in probes for use in the present invention, as the resulting probes have greater specificity than probes consisting entirely of DNA. Synthesis and uses of PNA have been disclosed by, for example, Orum et al, (1993 Nucl. Acids Res. 21, 5332); Egholm et al, (1992 J. Am. Chem. Soc. 114, 1895); and Egholm et al (1993 Nature 365, 566).
LNA is a synthetic nucleic acid analogue, incorporating xe2x80x9cinternally bridgedxe2x80x9d nucleoside analogues. Synthesis of LNA, and properties thereof, have been described by a number of authors: Nielsen et al, (1997 J. Chem. Soc. Perkin Trans. 1, 3423); Koshkin et al, (1998 Tetrahedron Letters 39, 4381); Singh and Wengel (1998 Chem. Commun. 1247); and Singh et al, (1998 Chem. Commun. 455). As with PNA, LNA exhibits greater thermal stability when paired with DNA, than do conventional DNA/DNA heteroduplexes. However, LNA can be synthesised on conventional nucleic acid synthesising machines, whereas PNA cannot; special linkers are required to join PNA to DNA, when forming a single stranded PNA/DNA chimera. In contrast, LNA can simply be joined to DNA molecules by conventional techniques. Therefore, in some respects, LNA is to be preferred over PNA, for use in probes in accordance with the present invention.
In particular, the target specific regions of the two probes may comprise LNA and/or PNA and the arm regions comprise DNA, with one or both of the probes comprising a destabilizing moiety. Chimeric probe molecules comprising PNA are useful only in those embodiments which do not require the copying of the PNA portions of a chimeric template, as PNA is not recognised as a template by any known nucleic acid polymerases.
It is an essential feature of the invention that the first and second probes, when hybridised to the target sequence, are adjacent or substantially adjacent to each other. Use of the term xe2x80x9cadjacentxe2x80x9d is herein intended to mean that there are no nucleotides of the target sequence left without base-pairing between those portions of the target sequence which are base-paired to the complementary sequence of the probes. This proximity between the probes enables the target-non-complementary sequences of the probes to anneal. As will readily be apparent to those skilled in the art, by designing the probes so as to allow for annealing to each other at greater separations from the target sequence, gaps may be introduced between the loci in the target nucleotide sequence to which the probes hybridise. In this situation the probes are said to be xe2x80x9csubstantially adjacentxe2x80x9d, because there may be some nucleotides of the target sequence left without base-pairing between those portions of the target sequence which are base-paired to the probes. Clearly, the number of intervening un-paired nucleotides of the target sequence can vary according to the design of the probes. Thus whilst it is preferred that the first and second probes hybridise so as to be adjacent, the probes may be separated by up to 5 nucleotides of target sequence, and the term xe2x80x9csubstantially adjacentxe2x80x9d is intended to refer to such situations.
In a second aspect the invention provides a method of detecting a nucleic acid target sequence of interest, the method comprising: hybridising a pair of probes in accordance with the first aspect defined above to the sequence of interest and to each other; causing extension of one of the probes using the other probe as template (e.g. as described in WO 93/06240 or U.S. Pat. No. 5,545,516 the contents of which are herein incorporated by reference), so as to form newly-synthesised nucleic acid; and detecting directly or indirectly the newly-synthesised nucleic acid. It is strongly preferred that the first probe is extended, using the second probe as a template, so as to form an active nucleic acid promoter, such that amplification can take place, e.g. by production of a large number of RNA copies of the second probe. Typically one or more further nucleic acid probes are introduced, in the presence of appropriate polymerases, so as to facilitate amplification. In preferred embodiments, a cycling amplification is established, which leads to multiple amplifications. Details of how such amplification may be obtained are driven in the examples below and in WO 93/06240.
Desirably the newly-synthesised nucleic acid, together with the template portion of the second probe will form an RNA polymerase promoter recognised, for example, by T3, T7 or SP6 RNA polymerases, or by any of the mutant forms thereof which are known to those skilled in the art. Particular mutant RNA polymerases are known, which may be useful in performing the method of the invention, which may synthesise RNA or DNA (see Kostyuk et al, 1995 FEBS Letts. 369, 165-168).
Thus, in preferred embodiments the arm region of the second probe (with or without destabilizing moiety) comprises a sequence complementary to the arm region of the first probe (+ or xe2x88x92 destabilizing moiety), and a unique sequence of choice such as, but not limited to, an RNA polymerase promoter sequence, a xe2x80x9c+12 regionxe2x80x9d to enhance efficiency of transcription, followed by probe detection and capture sequences.
By way of explanation, the present inventors have found that the efficiency of initiation of RNA synthesis by the RNA polymerase promoter is affected by sequences adjacent to the promoter, downstream. In particular, a region of twelve bases (the xe2x80x9c+12 regionxe2x80x9d) is required for optimum RNA transcription. It is therefore preferred that the template portion of the second probe, which is transcribed, comprises a +12 region appropriate to the polymerase which recognises the promoter. The inventors have elucidated the optimum sequence of +12 regions for the T7 polymerase (discussed in greater detail below)xe2x80x94it is not known at present if these are also optimum for say, T3 and SP6 polymerases. If, as is possible, SP6 and T3 polymerases have different optimum +12 regions, it would be a simple matter for the person skilled in the art to identify the relevant sequence by trial-and-error, with the benefit of the present disclosure.
The sequences of preferred +12 regions, for inclusion in the template portion of the promoter strand, (in respect of T7 polymerase) are shown below in Table 1. The most active +12 region (giving greatest transcription) is at the top, with the other sequences shown in decreasing order of preference.
Table 1 Alternative template +1 to +12 sequences for T7 polymerase, in descending order of transcription efficiency (Seq. ID Nos. 1-10 respectively)
5xe2x80x2 ATCGTCAGTCCC 3xe2x80x2
5xe2x80x2 GCTCTCTCTCCC 3xe2x80x2
5xe2x80x2 ATCCTCTCTCCC 3xe2x80x2
5xe2x80x2 GTTCTCTCTCCC 3xe2x80x2
5xe2x80x2 GATGTGTCTCCC 3xe2x80x2
5xe2x80x2 GTTGTGTCTCCC 3xe2x80x2
5xe2x80x2 ATCCTCGTGCCC 3xe2x80x2
5xe2x80x2 GCTCTCGTGCCC 3xe2x80x2
5xe2x80x2 GTTCTCGTGCCC 3xe2x80x2
5xe2x80x2 GTTGTGGTGCCC 3xe2x80x2
(The 5xe2x80x2 base is numbered as +1, being the first base downstream from the end of the promoter sequence, the 3xe2x80x2 base as +12).
In a further embodiment, the template portion of the complex (preferably on the promoter strand) could contain sequences that can be used to identify, detect or amplify the de novo synthesised RNA copies (see, for example, WO 93/06240. U.S. Pat. No. 5,554,516, or, for example, using molecular beacon sequences such as those disclosed by Tyagi and Kramer 1996 Nature Biotech 14, 303-308). These sequences are conveniently placed adjacent to, and downstream of, a +12 region (as described above) and may comprise, but are not limited to, one or more of the following: unique xe2x80x9cmolecular beaconxe2x80x9d sequences; capture sequences; detection probe complementary sequences: alternative RNA promoter sequences for use in an isothermal amplification cycling reaction (see below). A particular unique sequence especially useful in the present invention is provided by bases 791-820 of 16S ribosomal RNA from Streptomyces brasiliensis (Stackebrandt et al. 1991 Appl. Environ. Microbiol. 57, 1468-1477), which sequence has no alignment with any known human DNA or DNA of a known human pathogen.
In those embodiments where the invention involves the use of a mixture comprising both ribonucleotide triphosphates (for synthesis of RNA by an RNA polymerase) and dNTPs (for synthesis of DNA by a DNA polymerase) (e.g. where primer extension is followed by isothermal amplification), the concentration of dNTPs in the mixture will preferably not exceed 50 xcexcM, (preferably not exceed 10 xcexcm), as excessive concentrations of dNTPs have been found by the inventors to decrease the amount of RNA synthesised by the RNA polymerase.
In a particular embodiment, the invention provides a method of distinguishing between the presence of a sequence of interest and the presence of a closely-related variant thereof, which could differ from the sequence of interest by as little as one base (e.g. a point mutation). By selection of appropriate probe sequences, performance of the method of the invention can be made to produce very different results depending on whether the sequence present in the sample is the sequence of interest or a variant thereof. In particular, the presence of unpaired bases between the first probe and the target and/or between the second probe and the target, has been found to have a surprising effect on the amount of nucleic acid synthesised from the active promoter.
Generally, the inventors have found that design of the first probe to introduce a small number (e.g. 1-3) of bases unpaired with the sequence of interest, tends to reduce the amount of nucleic acid synthesised from the promoter. Conversely, and wholly unexpectedly, the inventors have found that the presence in the second probe of a small number (e.g. 1-3) of bases unpaired with the sequence of interest can decrease or increase the amount of nucleic acid synthesised from the promoter (the unpaired bases being near the xe2x80x9carmxe2x80x9d portion of the probe, such that the unpaired bases may be seen in some embodiments as a continuation of the target non complementary arm). The equivalent situation exists where there may be bases in the target sequence which are unpaired with the first probe (tending to cause a reduction in nucleic acid synthesis) or unpaired with the second probe (tending to have the opposite effect). In some embodiments, both the target and one or both probes may contain unpaired bases.
Without wishing to be bound by any particular theory, one hypothesis of the inventors is that the presence of unpaired bases between the second probe (which normally will also comprise the destabilizing moiety) and the target may, in some circumstances increase the flexibility of the resulting complex, thereby improving the access of bulky polymerase molecules to the promoter, and consequently increasing signal. In other circumstances the presence of unpaired bases can destabilize the interaction between the first and/or second probe and the target, thereby decreasing the amount of signal.
Thus, the inventors believe that inclusion of mismatches between the second probe and the sequence of interest should preferably be adjacent or substantially adjacent to the destabilizing moiety for optimum effect (i.e. preferably within 5 bases of the destabilizing moiety).
In a particular embodiment wherein the second probe, but not the first probe, comprises a destabilizing moiety (especially if the destabilizing moiety comprises a Hex dimer, as described below), the inventors have found that the presence of two adjacent unpaired bases in the second probe can increase the amount of nucleic acid produced from the promoter, but the presence of three unpaired bases can increase still further the amount of nucleic acid synthesised from the promoter.
In these embodiments the unpaired bases may be in the second probe, and may have counterpart unpaired bases in the sequence of interest (i.e. there are base mismatches). Alternatively, the bases may be unpaired because they are opposite a portion of the sequence of interest which comprises extraneous bases (present as a loop). Conversely, the unpaired bases may be present in the sequence of interest and the second probe comprises a loop of extraneous bases. Any variation from the sequence of interest which affects (increases or reduces) the number of unpaired bases in the second probe and/or the target sequence could in theory be detected although, as stated above, a variation from 1 to 2 (or vice versa) or 2 to 3 (or vice versa) in the number of unpaired bases is likely to give the greatest discrimination where the variant sequence differs by a single base from the sequence of interest. A greater number of variant bases will be more readily detected.
In a third aspect the invention provides a kit for detecting the presence of a nucleic acid target sequence of interest, the kit comprising a pair of probes in accordance with the first aspect and appropriate packaging means. The kit will typically be used for performing the method of the second aspect of the invention and conveniently comprise instructions for performing the method. The kit may advantageously comprise one or more of the following: a DNA and/or an RNA polymerase, labelling reagents, nucleotide triphosphates (labelled or otherwise), detection reagents (e.g. enzymes, molecular beacons) and buffers.
The destabilizing moiety is a chemical entity which is generally unable to undergo base pairing and hydrogen bonding in the normal manner as usually occurs when complementary strands of nucleic acid become hybridised. In the present invention the destabilising moieties effectively decrease the melting temperature (Tm) of the duplex which may be formed by the coming together of the two probes, such that in the presence of a third nucleic acid molecule (target) the molecules are able to form a much more thermodynamically stable three way junction. Hence, the presence of the destabilising moiety thermodynamically favours the three way junction over the relatively unstable probe duplex. Amplification of associated probes can then be achieved essentially as described, in detail. in WO 93/06240 (Cytocell Ltd). All manner of molecules may be suitable for use as a destabilizing moiety, although some compounds are specifically preferred, as described below. With the benefit of the present specification, the person skilled in the art will be able to test other compounds and readily select those which confer the appropriate degree of destabilization so as to prevent the hybridisation of probes in the absence of target nucleic acid of interest. Particularly preferred, as a matter of convenience, are those compounds which are commercially available in a form (e.g. as phosphoramidites) which facilitates their incorporation into synthetic oligonucleotides using conventional automated solid phase nucleic acid synthesisers.
Linker or spacer molecules have been used to introduce non-nucleotide segments into oligonucleotides. These molecules have been used to form folds and hairpins to bridge sections of oligonucleotides where no appropriate binding is possible, as well as simply to space tags further away from the oligonucleotide. A variety of such spacer molecules are available, many of which might be suitable for use as destabilizing moieties in the present invention. Such suitability could readily be ascertained by those skilled in the art with the benefit of the present disclosure.
In preferred embodiments, the first probe is such that the portion complementary to the sequence of interest (xe2x80x9ctarget specific regionxe2x80x9d or xe2x80x9cfootxe2x80x9d) is generally 10 bases or longer and the portion non-complementary to the sequence of interest (xe2x80x9carm regionxe2x80x9d) is generally 5 bases or longer. Generally, for the first probe, the target specific region will be longer than the arm region.
The second probe has a target specific foot region, also conveniently of xe2x89xa710 bases and an arm region conveniently of xe2x89xa720 bases. Generally, the arm region of the second probe will be longer than the complementary arm region of the first probe, such that the second probe arm region forms an xe2x80x9coverhandxe2x80x9d, which can act as a template for enzyme-mediated extension of the first probe in the presence of ribo- or deoxyribonucleotide triphosphates, for example as detailed in WO 93/06240. Thus, in a preferred embodiment, the 3xe2x80x2 end of the arm region of the first probe will desirably have a 3xe2x80x2 OH from which primer extension may be undertaken using the arm region of the second probe as template. The polymerase used to perform the extension will depend upon whether a thermal or isothermal reaction is sought. Preferably, the 3xe2x80x2 terminus of the second probe, when composed of DNA or RNA, should be blocked to prevent chain extension. It will be apparent to those skilled in the art how this could be achieved e.g. use of a 3xe2x80x2 phosphate, 3xe2x80x2 propyl or a 3xe2x80x2 dideoxynucleotide. The destabilizing moiety is typically located between the target specific region and the arm region, and may be present in the first probe and/or the second probe. Desirably the destabilizing moiety is present in the second probe. In certain applications, it may be desirable for the destabilizing moiety (additionally or alternatively) to be present in the arm region of the first probe. In some embodiments, the destabilizing moiety in one of the probes may lie partly opposite a portion of the target molecule, although this should normally be avoided.
The effects of the destabilizing moiety include: (a) reduction of background by destabilising hybridisation between the extension and template primer in the absence of target; (b) increasing target dependency through the improved control of background; and (c) release of steric compression at the three way junction and therefore assist access of polymerases. Destabilizing moieties which cannot base pair, but which nevertheless are capable of forming flexible folds and/or hairpin structures, are especially suitable. One such preferred destabilizing moiety comprises hexaethylene glycol (abbreviated herein as xe2x80x9cHexxe2x80x9d) (see FIG. 2), which may he present singly or in tandem up to n times (where n can be any number xe2x89xa71, but conveniently has a maximum value of 5). In a particularly preferred embodiment, the arm region of the second probe comprises two Hex molecules in tandem, where the number of bases opposite the destabilising moiety in the arm region of the first probe should be six to eight bases (most preferably six), followed by a complementary region, preferably of 5-15 bases. An alternative preferred destabilizing moiety comprises a plurality of alkylene (especially methylene) repeats. Particularly preferred are penta- or hexa-methylene spacers.
Other, less preferred, destabilizing moieties may alternatively be used. These include, but are not limited to, inosine, Virazole(trademark) (N[1]-[1-xcex2-D ribofuranosyl] 3-carboxamido-1,2,4,-triazole), Nebularin(trademark) (N[9]-[1-xcex2-D ribofuranosyl]-purine), nitropyrrole, ribose, propyl or combinations of the above eg. propyl-Hex-propyl, propyl-Hex-Hex-propyl, etc. Propyl may be replaced by, for example, ethyl, butyl, pentyl, heptyl, octyl etc. The number of bases opposite the destabilizing moiety in the arm region of the reciprocal probe should be x, where x is xe2x89xa71. The exact number of bases will of course depend on the size of the destabilizing moiety and the value of n.
The following may be used as a guide: for each Hex molecule in the destabilizing moiety, the opposite oligonucleotide should preferably comprise 3-4 bases (preferably 3); for each other molecule or radical mentioned above present in the destabilizing moiety, the opposite oligonucleotide should preferably comprise a single base, with the exception of the following: butylxe2x80x94two bases, pentylxe2x80x94two bases, heptylxe2x80x94three bases, and octylxe2x80x94four bases.
The chemicals described above and used as destabilizing moieties are all commercially available (e.g. from Glen Research, USA).
In a further embodiment of the invention it may be advantageous, when seeking to detect a sequence of interest in a mixture comprising double stranded DNA (such as genomic DNA), to include in the hybridisation mixture further oligonucleotides (xe2x80x9cblocking oligonucleotidesxe2x80x9d). These blocking oligonucleotides hybridise to the sequence of interest on either side of the portion which is complementary to the first probe and the portion complementary to the second probe. The blocking oligonucleotides preferably comprise DNA, PNA, LNA (or a combination thereof) and advantageously each comprise at least 10 (more preferably at least 20) nucleotides. The purpose of the blocking oligonucleotides is to inhibit (under the hybridisation conditions employed) re-annealing of the target strand with its complementary strand. The blocking oligonucleotides may anneal to the target strand substantially adjacent to the first and second probes, or may anneal at a distance (e.g. 5-50 bases) therefrom.
Blocking oligonucleotides may offer little advantage if the first and/or second probes contain large target-complementary xe2x80x9cfeetxe2x80x9d regions.
As mentioned above, the formation of a three way junction in accordance with the method of the invention will typically result in the de novo synthesis of nucleic acid, normally RNA. The newly-synthesised nucleic acid may be detected directly or indirectly by any of a number of techniques, preferably following an amplification step. Further details of suitable detection and amplification processes are given below.
Detection Methods
Nucleic acid produced from a three way junction in accordance with the method of the invention could be detected in a number of ways, preferably following amplification (most preferably by means of an isothermal amplification step). For example, newly-synthesised RNA could be detected in a conventional manner (e.g. by gel electrophoresis), with or without incorporation of labelled bases during the synthesis.
Alternatively, for example, newly-synthesised RNA could be captured at a solid surface (e.g. on a bead, or in i microtire plate), and the captured molecule detected by hybridisation with a labelled nucleic acid probe (e.g. radio-labelled, or more preferably labelled with an enzyme, chromophore, fluorophore and the like).
One preferred detection method involves the use of molecular beacons or the techniques of fluorescence resonance energy transfer (xe2x80x9cFRETxe2x80x9d), delayed fluorescence energy transfer (xe2x80x9cDEFRETxe2x80x9d) or homogeneous time-resolved fluorescence (xe2x80x9cHTRFxe2x80x9d). Molecular beacons are molecules which it fluorescence signal may or may not be generated, depending on the conformation of the molecule. Typically, one part of the molecule will comprise a fluorophore, and another part of the molecule will comprise a xe2x80x9cquencherxe2x80x9d to quench fluorescence from the fluorophore. Thus, when the conformation of the molecule is such that the fluorophore and quencher are in close proximity, the molecular beacon does not fluoresce, but when the fluorophore and the quencher are relatively widely-separated, the molecule does fluoresce. The molecular beacon conveniently comprises a nucleic acid molecule labelled with an appropriate fluorophore and quencher.
One manner in which the conformation of the molecular beacon can be altered is by hybridisation to a nucleic acid, for example inducing looping out of parts of the molecular beacon. Alternatively, the molecular beacon may initially be in a hair-pin type structure (stabilised by self-complementary base-pairing), which structure is altered by hybridisation, or by cleavage by an enzyme or ribozyme.
FRET (Fluorescence Resonance Energy Transfer) occurs when a fluorescent donor molecule transfers energy via a nonradiative dipolexe2x80x94dipole interaction to an acceptor molecule. Upon energy transfer, which depends on the Rxe2x88x926 distance between the donor and acceptor, the donor""s lifetime and quantum yield are reduced and the acceptor fluorescence is increased or sensitised.
The inventors have used FAM (6-carboxyfluorescein) and TAMRA (N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-6-carboxy rhodamine) as donor and acceptor in a nucleic acid hybridisation assay. The assay uses two dye labelled DNA oligomers (15 mers). FAM is linked to the 5xe2x80x2 of one probe and TAMRA to the 3xe2x80x2 of the other. When hybridised to target nucleic acid the probes are positioned adjacent to one another and FRET can occur. The inventors"" experiments have demonstrated that for maximum signal the probes need to be spaced by five bases. Optimum spacing for DEFRET and HTRF (discussed below) may be different (often less).
Another approach (DEFRET, Delayed Fluorescence Energy Transfer) has been to exploit the unique properties of certain metal ions (Lanthanides e.g. Europium) that can exhibit efficient long lived emission when raised to their excited states (xcexexcitation=337 nm, xcexemission=620 nm). The advantage of such long lived emission is the ability to use time resolved (TR) techniques in which measurement of the emission is started after an initial pause, so allowing all the background fluorescence and light scattering to dissipate. CY5 (xcexexcitation=620 nm, xcexemission=665 nm) can be used as the DEFRET partner.
HTRF (see WO 92/01224 and U.S. Pat. No. 5,534,622) occurs where the donor (Europium) is encapsulated in a protective cage (cryptate) and attached to the 5xe2x80x2 end of an oligomer. The acceptor molecule that has been developed for this system is a protein fluoropohore, called XL665. This molecule is linked to the 3xe2x80x2 end of a second probe. This system has been developed by Packard.
In another embodiment, the newly-synthesised RNA, before or after amplification, results in formation of a ribozyme, which can be detected by cleavage of a particular nucleic acid substrate sequence (e.g. cleavage of a fluorophore/quencher-labelled oligonucleotide).
Amplification Techniques
In preferred embodiments of the present invention, the RNA derived from the target dependent transcription reaction is amplified prior to detection, the amplification step typically requiring the introduction of a DNA oligonucleotide. The amplification step is advantageously effected isothermally (i.e. without requiring thermal cycling of the sort essential in performing PCR). The introduced DNA oligonucleotide is complementary to the 3xe2x80x2 region of the newly synthesised RNA and also contains the sequence of an RNA polymerase promoter and a unique transcribable sequence (template portion). Upon hybridisation of the newly-synthesised RNA with the DNA oligonucleotide, a primer extension reaction from the 3xe2x80x2 end of the RNA, mediated by an added DNA polymerase, produces a functional double stranded RNA polymerase promoter. In the presence of the relevant RNA polymerase, multiple copies of a second RNA species are synthesised from the unique region of the DNA oligonucleotide. This RNA in turn can act as primer to a further round of primer extension and RNA synthesis. The synthesis of further RNA requires the presence of another DNA oligonucleotide that is complementary to the 3xe2x80x2 region of the second RNA species. This DNA oligonucleotide also contains the sequence of an RNA polymerase promoter element together with a sequence upon transcription of which produces RNA identical to that derived in the target dependent transcription reaction. The 3xe2x80x2 end of the RNA thus synthesised is complementary to the first DNA oligonucleotide and hence a cyclical amplification system is generated.
In a variant of the embodiment described above, the introduced DNA oligonucleotide hybridises to the de novo synthesised RNA, the respective sequences being such that a further RNA polymerase promoter is directly formed without the need for a DNA polymerase-mediated extension step. A cycling reaction may then be performed essentially as described above, with the transcript from one reaction hybridising with a DNA oligonucleotide to form a second RNA promoter, which produces a transcript having the same sequence as the original transcript.
In the above amplification strategies some background xe2x80x9cnoisexe2x80x9d may be created because of the tendency of many RNA polymerases (at relatively low frequency) to produce RNA transcripts of a single stranded DNA sequence such that, for example, some transcription of single stranded DNA oligonucleotides may occur even in the absence of appropriate complementary strands. It is possible that this low level of background transcription can be reduced by designing the DNA oligonucleotides so as to incorporate near their 3xe2x80x2 end a sequence which tends to cause termination of transcription. One example of such a sequence, which is especially effective at terminating T7 polymerase-mediated transcription, is AACAGAT (in the template strand), as disclosed by He et al. (1998 J. Biol. Chem. 273, 18,802). The same or a similar termination sequence could be positioned at the 5xe2x80x2 end of the DNA template to increase processivity.